Oscillating microtome with flexure drive

ABSTRACT

A microtome method and apparatus includes a microtome blade configured to oscillate in a direction transverse to a direction of advancing a cut, and a first flexure to support and guide the blade. The first flexure is compliant in the transverse direction while being stiff in the cut direction. A second flexure operatively engaged at one end portion with the first flexure, is stiff in the transverse direction while being compliant in the cut direction. The other end portion of the second flexure is rotatably engaged by an eccentric driven by a rotatable actuator, which oscillates the blade in the transverse direction while effectively isolating non-transverse motion from the blade. The second flexure is configured to move independently of any guides or other stationary objects during oscillation.

This application is a Continuation of U.S. patent application Ser. No.13/166,472, entitled Oscillating Microtome with Flexure Drive, filed onJun. 22, 2011, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/357,896, entitled Drive for an Oscillating Microtome, filedon Jun. 23, 2010.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

Embodiments of the present invention relates generally to a microtomehaving an oscillating blade. More particularly, this invention relatesto a microtome having flexure drive in the form of a pair of compliantmechanisms having substantially orthogonal axes of predominantstiffness, configured to oscillate a blade transversely to a directionin which the cut advances.

(2) Background Information

A microtome is a sectioning instrument that allows for the cutting ofthin slices of material known as sections. Microtomes are an importantdevice in microscopy preparation, allowing for the preparation ofsections for observation under transmitted light in optical microscopy,or in block face imaging, a microtome may be used to successively removeportions of the sample to expose the sample interior for imaging of theremaining specimen block. To section a sample, a blade similar to arazor blade is used which is drawn across the sample thus removing athin slice of the specimen under consideration. Microtomes generally usesteel, glass, or diamond blades depending upon the specimen being slicedand the desired thickness of the sections being cut. Steel blades aretypically used to prepare sections of animal or plant tissues for lightmicroscopy histology.

Oscillating blade microtomes are a variation of the basic microtome, andare widely recognized as superior for cutting thick sections fromnon-embedded or fresh tissue samples. The vibration amplitude, thevibrating speed, the angle of the blade, and the feed rate (of tissuesamples) may all be controlled allowing for optimization of the cuttingprocess. Fixed or fresh tissue pieces are typically embedded in lowgelling-temperature agarose. The resulting agarose block containing thetissue piece is then glued to a stand and sectioned while submerged in awater or buffer bath.

Examples of conventional oscillating microtomes are disclosed in U.S.Pat. No. 6,651,538, entitled Microtome (the '538 patent), and U.S. Pat.No. 6,041,686, entitled Microtome Having an Oscillating Blade (the '686patent). In the '538 patent, blade oscillation is provided by attachingthe blade to a movable body that is coupled to a base by a resilientcoupler. The base is provided with a driving electromagnet, and themovable body is provided with a permanent magnet. The movable body isthen oscillated by supplying a control signal to the drivingelectromagnet. The '686 patent discloses an articulated connectioncoupled to an eccentric that converts the circular motion of an electricmotor into an oscillating, substantially linear motion. These approacheseach suffer from disadvantages associated with relatively impreciseoscillatory motion, including parasitic error motions, which tend toresult in imprecise section thicknesses and tissue damage.

The '686 patent and similar motorized configurations may offer improvedcutting accuracy relative to the directly-driven solenoid approaches ofthe '538 patent and the like. However, such motorized approaches sufferfrom disadvantages due to backlash inherent in the joint of thearticulated arm. A joint is not perfectly stiff, and the magnitude ofthe backlash may be on the order of tens of microns to several hundredmicrons, introducing both higher order harmonics into the motion of theblade, and parasitic motion along orthogonal axes.

Drawbacks are also associated with the alternate approach disclosed inthe '686 patent, of using a guide rail to guide the motion of a leafspring connected to the blade assembly. In this alternate approach, aswell as the articulated arm approach discussed above, the guide raildisadvantageously limits the upper frequency and magnitude of theoscillations due to friction between the oscillating spring/arm and thestationary rail. Furthermore, again due to backlash or imperfections ofthe guide rail, it is difficult, if not impossible, to eliminateunwanted motions perpendicular to the blade motion.

Another issue is potential buckling of the leaf spring. Any bucklingwould tend to reduce the stiffness along the desired direction ofmotion. Moreover, the friction from the guide rail generally leads toinstrument wear and required maintenance.

There is therefore a need for an improved microtome that addresses thedrawbacks of the prior art.

SUMMARY

In one embodiment of the present invention, a microtome apparatusincludes a microtome blade configured to oscillate in a directiontransverse to a direction of advancing a cut, and a first flexureconfigured to support and guide the blade, the first flexure configuredto be compliant in the transverse direction while being stiff in the cutdirection. A second flexure operatively engaged at one end portion withthe first flexure, is configured for being stiff in the transversedirection while being compliant in the cut direction. The other endportion of the second flexure is rotatably engaged by an eccentricdriven by a rotatable actuator, which oscillates the blade in thetransverse direction while effectively isolating non-transverse motionfrom the blade. The second flexure is configured to move independentlyof any guides or other stationary objects during the oscillation.

In another aspect of the invention, a tissue scanning imaging apparatusincludes the foregoing embodiment, an imaging device; and a stageconfigured to selectively advance a specimen sample into operativeengagement with both the microtome apparatus and the imaging device.

Yet another aspect of the invention includes a method of treating asample with the above-described microtome embodiment. This methodincludes oscillating the microtome blade in a direction transverse to adirection of advancing a cut, and moving the sample in the cut directioninto operative engagement with the oscillating microtome blade to removea section from the sample.

The features and advantages described herein are not all-inclusive and,in particular, many additional features and advantages will be apparentto one of ordinary skill in the art in view of the drawings,specification, and claims. Moreover, it should be noted that thelanguage used in the specification has been principally selected forreadability and instructional purposes, and not to limit the scope ofthe inventive subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example and notlimitation in the figures of the accompanying drawings, in which likereferences indicate similar elements and in which:

FIG. 1 is an elevational, schematic view of a sample being sectioned inaccordance with aspects of the present invention;

FIG. 2 is a block diagrammatic plan view of aspects of the presentinvention;

FIG. 3A is a schematic plan view of an embodiment of the presentinvention;

FIG. 3B is a perspective view of elements of the embodiment of FIG. 3;

FIG. 4A is a perspective view of the embodiment of FIG. 3A;

FIGS. 4B and 4C are top and bottom perspective views of elements of theembodiment of FIG. 4A;

FIG. 4D is a schematic elevational view illustrating aspects of theembodiments of FIGS. 2-4C;

FIG. 5 is a schematic elevational view of a representative applicationincorporating the embodiments of FIGS. 2-4D; and

FIG. 6 is a schematic plan view of an alternate embodiment of thepresent invention.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings that form a part hereof, and in which is shown byway of illustration, specific embodiments in which the invention may bepracticed. These embodiments are described in sufficient detail toenable those skilled in the art to practice the invention, and it is tobe understood that other embodiments may be utilized. It is also to beunderstood that structural, procedural and system changes may be madewithout departing from the spirit and scope of the present invention. Inaddition, well-known structures, circuits and techniques have not beenshown in detail in order not to obscure the understanding of thisdescription. The following detailed description is, therefore, not to betaken in a limiting sense, and the scope of the present invention isdefined by the appended claims and their equivalents.

Embodiments of the present invention are directed to microtomes whichemploy an oscillating blade to cut a specimen into sections. A primaryflexure mechanism guides the motion of a blade, while being coupled toan oscillating actuator via a secondary flexure mechanism. The additionof a secondary flexure eliminates or significantly reduces unwantedmechanical coupling between the actuator and the first flexure,diminishing undesirable vibrations along directions nonparallel to thedesired oscillatory blade motion. In particular, reduced vibrations inthe out-of-plane direction (z-axis as shown and described herein)perpendicular to the surface of the specimen tend to reduce damage tothe specimen during the cutting process. Moreover, the second flexureoperates in a substantially frictionless manner, e.g., without the usefriction-inducing guide rails.

In comparison to conventional oscillating microtome configurations, lesswaviness and damage to the section may be realized, allowing thinnersections and better quality imaging. This may be particularly useful forimaging sections and for block face imaging into the specimen blocksurface for microscope or other imaging investigations. Furtheradvantages of this approach may include higher speed operation, betterrepeatability, lower cost, higher robustness, easier manufacturability,greater design flexibility, and longer instrument life, relative toconventional approaches.

Where used in this disclosure, the terms x, y or z “-axis” and/or“-direction” refer to one of three directions in the three dimensionalorthogonal Cartesian coordinate system illustrated in FIGS. 2, 3A and3B. The terms “cut direction” and/or “direction of advancing a cut”refer to the movement along the y-axis in the illustrated coordinatesystem. The term “transverse” refers to a direction other thansubstantially parallel to the cut direction, such as along the x-axis inthe illustrated coordinate system.

An aspect of the present invention was the recognition that compliantmechanisms, also known as flexures, which have conventionally been usedfor controlled motion in very small increments (e.g., with translationalresolution of nanometer increments, and rotational resolution of microradians), may be successfully employed in the larger scale, high speedblade oscillation of microtomes.

Flexures are mechanisms that include a series of rigid bodies connectedby integral compliant elements to produce a geometrically well-definedmotion upon application of a force. The present inventors recognizedthat flexures offer many potential benefits: they are relatively simpleand inexpensive to manufacture and assemble; unless fatigue cracksdevelop, the flexures undergo no irreversible deformation (elasticdeformation) and are therefore wear free; complete mechanisms may beproduced from a single monolith; mechanical leverage is easilyimplemented; displacements are smooth and continuous even at thenanometer level of motion; failure mechanics such as fatigue and yieldare well understood; they may be designed to be insensitive to thermalvariation and mechanical disturbance (vibrations); symmetric designs maybe inherently compensated and balanced; there is a linear relationshipbetween applied force and displacement for small distortions; forelastic distortions, this linear relationship is independent ofmanufacturing tolerance, though the direction of motion will be lessaccurate as these tolerances are relaxed; and, flexures have atomiclevel of repeatability. Examples of such flexures/compliant mechanismsare disclosed in U.S. Pat. Nos. 7,093,827 and 7,270,319, both entitledMultiple Degree of Freedom Compliant Mechanism, to Martin L. Culpepper,and which patents are incorporated herein by reference for all purposes.

The present inventors further recognized that properly designed flexuresallow relatively high accuracy motion control, even allowing errors dueto manufacturing tolerances to be corrected. In comparison, moretraditional mechanisms not based on flexures, such as the articulatedlinkages discussed hereinabove, tend to exhibit backlash, which may bedifficult to control even in closed loop systems. This ultimately lowerstheir precision in comparison to flexure mechanisms. Flexures may thusbe used to help achieve at least two goals: (1) providing a precisedisplacement upon application of a specific applied force, and (2)providing a precisely known force upon application of a specific applieddisplacement. (Flexures may also be used for motionamplification/deamplification, i.e. providing a precisely knowndisplacement upon application of a specific applied displacement.)

As shown and described herein, the flexure coupling(s) of the presentinvention substantially eliminates unwanted parasitic motions withoutthe need for a separate guide rail or the use of an articulated arm,while still maintaining relatively high stiffness or rigidity in desireddirections. Embodiments of the present invention may substantiallyreduce cost, complexity, and wear, while improving instrumentperformance, robustness and manufacturability. The flexures effectivelyform high performance mechanical bearings which allow motion by thebending of a load element. Since they are constructed from a monolithicmaterial (e.g., a single piece of metal), they have no internal movingparts, to provide excellent wear characteristics, and precise andrepeatable ranges of motion.

In general, motion control requires two separate functional units: aforce generating unit to actuate the motion and a bearing unit toconstrain and guide the motion to the desired trajectory. Flexurebearings use elastic deformation of materials to generate degrees offreedom to allow some motions while minimizing others. The allowedmotions are determined by the geometry cut into the material of thebearing.

For an oscillating microtome, as mentioned above, motion in the out ofplane (z-direction as shown) may result in poor quality sectioning. Theflexure stages shown and described herein may effectively minimize anysuch parasitic z-axis motions, providing a peak-to-peak amplitude on theorder of 1 to 2 μm for a sample surface flatness of <1 μm at oscillationfrequencies of 200 Hz or more.

Referring now the appended figures, exemplary embodiments of the presentinvention will be described in detail. Turning to FIG. 1, the volume ofmaterial removed per section(s) when using a microtome of the presentinvention, is given by the following Equation 1:V _(Removed) =d*W*V _(T)  Eq. 1

V_(T) is the feed rate of the blade relative to the sample, d is thethickness of the section, W is the width of the tissue section sample, αis the tool (blade) cutting angle; F is the vibration (oscillation)frequency of the blade motion (along the x-axis in the coordinate systemshown and described herein), and A is the amplitude of the blade motion.

As will be discussed in greater detail hereinbelow, the frequency andamplitude of the blade may be adjusted depending on the thickness of thetissue section, the feed rate, the tool angle, and the materialproperties of the tissue itself. In practice, good quality sections overa wide range of section thickness may be achieved by adjusting thecutting angle, feed rate and vibration frequency.

Turning now to FIG. 2, a microtome 20 (which may also be referred to asa microtome with a vibrating or oscillating blade) of the presentinvention uses a second flexure mechanism F2 in lieu of a conventionalarticulated linkage between an actuator (e.g., motor) 22 and the bladeassembly 24. The flexure F2 is rigidly mounted to another motionguidance flexure mechanism shown as first flexure F1. The second flexureF2 is configured to decouple unwanted motions between the actuator andthe blade assembly flexure F1. The flexure F2 may take any number ofconfigurations that will be evident to those skilled in the art in viewof the instant specification. Examples include, but are not limited to,folded beam, shape morphing designs, and bow flexures. Unlike theabove-described mechanical linkages of the prior art, flexure F2 isstiff along the direction of the desired motion (i.e., along the x-axisas shown), and flexible in a direction orthogonal thereto (e.g., they-direction as shown), without suffering from buckling.

As also shown, actuator 22 provides the oscillatory force to drive theblade. Particular embodiments of the actuator 22 include but are notlimited to, a rotating electric motor with an offset cam or othereccentric, an eccentric drive, a solenoid, and an electromagnet.

The flexure F1 guides the motion of the blade, typically an oscillatorymotion along the x-axis. Particular embodiments of F1 guide the bladealong an arcuate path as this has been shown to improve section qualityin some applications.

Thus, in particular embodiments, flexure F2 is linearly and rotationallyconstrained along x, z, and θy, and is not constrained along y,θx, orθz. Flexure F1, to which the blade assembly is rigidly attached, isconstrained along y, z, θx, θy, and θz, and is not constrained along x.

The aforementioned stiffness in the x-direction, and compliance in otherdirections, enables flexure F2 to help maintain parasitic motion,particularly force transmitted in the out of plane (z-axis) direction asa relatively small fraction of the motion and force along the x-axis.Typical ratios exhibited by the embodiments shown and described hereinmay be on the order 0.0001 or one part in 10,000. It is also noted thatthe compliance of flexure F2 may compensate for various misalignments,such as may be due to imprecise mounting of the actuator 22. That is,position and angle errors along one or more of its unconstrained degreesof freedom may be effectively decoupled by its compliance, whiletransmitting motion in the x-direction. This helps to facilitaterelatively precise motion control.

These configurations provide for a relatively small overall systemfootprint, and tend to be superior to prior art approaches, e.g., interms of size, performance and/or cost. This use of flexure F2 alsoprovides flexibility to tailor the dynamics of the driving force to theoscillating blade.

Turning now to FIGS. 3A and 3B, a representative embodiment of amicrotome of the present invention is shown as 20′. The blade holderassembly 24′ is connected to a mounting platform (ground) 28 through aflexure arm F1′ which allows motion along the x-axis. The blade holderassembly 24′ is actuated via flexure F2′ connected to an actuator 22′ inthe form of an electric (e.g., DC) motor (e.g., a Faulhaber 3863H012C,by Faulhaber). The motor 22′ is connected to the flexure F2′ by use ofan eccentric 30, e.g., in the form off center cam as shown. Cams withdifferent offsets may be used to adjust the amplitude of the oscillatorymotion. As described above, the flexure linkage F2′ transforms therotary motion of the DC motor 22′ to a harmonic linear motion with afrequency corresponding to the rotational speed of the motor 22′. Asalso discussed above, the flexure linkage F2′ minimize any forcestransmitted to the blade holder assembly 24′ which are not co-linearwith the desired linear motion (e.g., along the x-axis) of the blade.

In the particular embodiment shown, flexure F2′ includes a series offlexible beams 29 spaced from one another in the cut direction. In theexample shown, flexure F2′ is provided with a relatively high resistanceto buckling by use of an aspect ratio (i.e., length L to width W, FIG.3A) which is low relative to that of a conventional leaf spring. Thisuse of parallel beams, with one or more connectors 31 therebetween,provides the overall flexure F2′ with both relatively high stiffness inthe x-direction to prevent buckling, while still providing relativelyhigh flexibility in a direction (e.g., y-direction) orthogonal thereto.It should also be noted that these aspects enable flexure F2′ (and F2″,FIG. 6) to move in a substantially frictionless manner duringoscillation. This contrasts with conventional articulated linkages andleaf spring approaches which rely on engagement with a substantiallystationary guide rails in order to maintain the desired motion.

As shown, beams 29 extend from the one end portion towards the other endportion of flexure F2′, and in the particular embodiment shown, extendsubstantially parallel to one another. In this regard, it should benoted that beams that are bowed, curved, and/or with varyingcross-sectional area when at rest, such as the alternate beams shown inphantom at 29′ in FIG. 3A, would still be considered to be substantiallyparallel with one another. In this regard, as mentioned above, bowedbeams, or beams having substantially any other desired shaped, may beused without departing from the scope of the present invention.

In particular embodiments, the flexible beams 29, 29′, etc., areconfigured to extend in the transverse direction (e.g., x-direction asshown) at some point (e.g., the midpoint) of the oscillation cycle asshown. Moreover, it should be recognized that during the oscillation,the one end portion of the second flexure F2′ connected to the firstflexure F1′, is configured to move in the transverse (e.g., x)direction, while being substantially prevented from moving in the cut(e.g., y) direction, while the other end portion connected to actuator22′, is configured to move in both the transverse and cut directions.

As also shown, particular ones of the beams 29, 29′ are connected to oneanother with connectors 31 disposed at spaced locations along the lengthof flexure F2′. In particular embodiments, at least one connector 31 isdisposed within approximately the middle 50 percent (%) of the length Lof the flexure F2′.

The frequency of the cutting motion may be set by adjusting the speed ofthe motor 22′. The amplitude of the motion may be specified by adjustingthe offset of the cam 30, as discussed above, e.g., either by replacingthe cam 30 with one having a different offset, or by employing a camhaving an adjustable offset, as will be familiar to those skilled in theart.

A blade 32 may be fastened to the blade holder assembly 24′ in anyconvenient manner, such as by clamping using a micro flexure. The bladeangle α (FIG. 1A) may be manually adjusted by rotating the blade holderand locking it to the blade holder assembly, as will be discussed ingreater detail hereinbelow. As a non-limiting example, the blade 32 is a2.5″×0.3″ carbon steel blade of the type commonly used in histology. TheF1′ and F2′ flexures may be manufactured out of, for example, Aluminum(e.g., Aluminum 7075 or 6061), titanium, spring steel, and/or silicon.Electrical discharge machining or a water jet may be used to manufactureany one or more (or all) of flexure F1′, flexure F2′, and mountingplatform 28, from a single monolithic piece of material as shown in FIG.3B. In this regard, In general, any material having a relatively highyield strength to elastic modulus ratio may be used.

With reference now to FIGS. 4A-4C, aspects of microtome 20′ will bedescribed in greater detail. Turning to FIG. 4A, microtome 20′ is shownwith actuator (motor) 22′ and blade holder 24′ disposed in operationalengagement with flexure F1′. Flexure F2′ is connected at one end toflexure F1′, and at the other end to motor 22′. The connection andrelative orientation of flexure F2′ with motor 22′ may be accomplishedin any convenient manner. For example, a connector 40 may be used, whichconnects via set screw to the flexure F2′ at one end, while rotatablyengaging a dowel 42 extending from offset cam 30 (FIG. 3A) at the otherend. The offset cam is mounted to the shaft of the motor 22′.

The blade holder assembly 24′ has a number of unique features, whichwill now be described with reference to FIGS. 4B-4D.

Turning now to FIGS. 4B and 4C, embodiments of the blade holder mayinclude a blade clamping mechanism including an upper clamp 34 and alower clamp 36. Small magnets (not shown), e.g. neodymium magnets, maybe receivably disposed within receptacles 38 and 40 of clamps 34 and 36,respectively, to provide a compressive force capable of securelyfastening the blade 32 to the blade holder assembly 24′. The upper clamp34 may also be provided with a curved surface (not shown) configured toallow the tissue section to slide easily on the blade holder while it isbeing sectioned as shown in FIG. 1A.

As also shown, the blade holder 24′ may be attached in a variable mannerto flexure F1′ to facilitate changing angle α (FIG. 1) that the blade 32makes with respect to the tissue to be sampled. In the embodiment shown,angle α may be adjusted by loosening the holder arm clamps 44 to releasethe arm rod 46 to permit the blade holder arm 48 to be pivotedthereabout. Once set at the desired angle, the blade holder arm 48 maybe secured in place by tightening the holder arm clamps 44.

In addition, provision may be included to adjust the blade 32 in thez-direction, to permit precise level adjustment of the blade 32 relativeto the tissue being cut. For example, such adjustment may be providedset screws 50 disposed to adjust the position along the z-axis of theblade holder arm rods 46 prior to being secured by claims 44.

Thus, as shown and described, blade holder 24′ is relativelylightweight, to enable increased frequency and amplitude of the blademotion relative to many prior art approaches. The magnetic clampingsystem provides for a relatively low mass to force ratio. The magneticclamping mechanism is also easy to use and does not require the use oftools to remove and replace the blade 32. It offers high reproducibilityand the entire assembly may be easily manufactured, such as by usingconventional 3D printing technology. This tends to reduce cost andincrease design flexibility.

Turning now to FIG. 4D, this lightweight approach also enables thecenter of stiffness of the blade holder 24′ (with respect to motion inthe θy direction) to be located relatively close to the x-axis, which inthe embodiments shown, passes through the geometric center of flexureF2′ (and F2″, FIG. 6). Indeed, in some embodiments, a mass 52 may bedisposed on the blade holder 24′ on a side opposite that of the blade32, which is sized to bring the center of stiffness to the x-axis asshown at 53. This approach helps to reduce, if not substantiallyeliminate, any parasitic swing motion (shown at 54) of the blade holder24′ about the y-axis, which would tend to generate parasitic errormotion in the z-direction during oscillation. It should also berecognized that any Ox error motion (i.e., rotation about the x-axis),which would also generate parasitic error motion along the z-axis, isalso reduced by the relatively low mass of the blade holder 24′ as shownand described herein. These features of blade holder 24′, in combinationwith the use of flexures F1, F1′ and F2, F2′, F2″ as shown and describedherein, have been shown to reduce any parasitic z-motion of the bladeholder 24′ to between 0.5 and 1.0 micron.

Turning now to FIG. 5, as shown schematically, microtome 20, 20′, etc.,may be incorporated within an imaging system to effect both sectioningand imaging of a sample. For example, in an exemplary embodiment, thesample may be placed within a bath (e.g., water bath) 55 which may bemoved along the cutting axis (e.g., along the x-axis, FIG. 2) relativeto microtome 20, 20′, etc., for sectioning, and an imaging device suchas a microscope 56, for imaging. Substantially any microscope or otherimaging device including conventional CCD and/or laser scanning PMT(Photomultiplier tube)-based devices may be used. In this regard,examples of both imaging (i.e., microscope) systems and methodologieswith which the microtomes 20, 20′, etc., of the instant invention may beused, are disclosed in U.S. Pat. No. 7,372,985, entitled Systems andMethods for Volumetric Tissue Scanning Microscopy, which patent is fullyincorporated herein for all purposes.

Turning now to FIG. 6, an alternate embodiment of the present inventionis substantially similar to the embodiment shown and described in FIGS.3A-4C, but for the use of an alternative second flexure F2″. Thisflexure F2″ uses dual, parallel beams 29 with a single connector 31disposed within the middle 50 percent of the length of the flexure F2″,e.g., at the midpoint of the longitudinal dimension of the flexure F2″.This particular embodiment uses only a single connector 31 within thismiddle 50 percent, though a plurality of connectors disposed in spacedrelation along the beams 29 may also be used, if desired to provideadditional resistance to buckling. This construction provides a smallerfootprint than flexure F2′, and a less complex dual-beam construction,which tends to lower the moving mass and simplify construction forcomparatively higher frequency operation and lower parasitic motion inmany applications.

Referring back to FIG. 2, flexures F2, F2′, F2″, etc., have been shownto provide a high ratio of x-direction stiffness (K_(X2)) to y-directionstiffness (K_(Y2)), for efficient error motion decoupling. Examples offlexure F2″ in particular, fabricated from aluminum, have been shown toprovide a ratio K_(X2)/K_(Y2) of at least approximately 500, and inparticular embodiments, more than 2500.

It is noted that microtome 20 (and 20′, 20″, etc.) will have a resonancefrequency that may vary depending upon the particular parameters (e.g.,stiffness) of the flexures F1, F1′, and F2, F2′, F2″, etc., used in anyparticular embodiment. In general, the stiffer the flexures (e.g., inboth the x and y directions), the higher the resonance frequency of themicrotome system. Embodiments of the present invention may be providedwith a resonance frequency that is at least about 2 to 4 times thehighest desired operational speed of the microtome, to ensure smoothoperation of the microtome.

It should be recognized that using a flexure that is excessivelycompliant in the y direction (i.e., has a very low K_(Y2)) may make iteasy to achieve the relatively high ratio K_(X2)/K_(Y2) as indicatedabove. However, such a low K_(Y2) may provide the microtome with aresonance frequency that will be too low for some applications. Thus,the skilled artisan will recognize, in light of the teachings hereof,that embodiments of the present invention may be provided with a balancebetween the ratio K_(X2)/K_(Y2) and the resonance frequency of thesystem, to achieve desired performance at particular operational speeds.

Particular examples of microtome 20, 20′, 20″, have a resonancefrequency of at least 500 Hz. Similarly, in many applications it may bedesirable for embodiments of the invention to have an out-of-plane(z-axis) mode shape at a frequency that is at least about 4 to 8 timesthe highest desired operational speed of the microtome, to help preventthe occurrence of unwanted parasitic motion. Particular examples ofmicrotome 20, 20′, 20″, etc., have been shown to have a z-axis modeshape frequency of at least 700 Hz.

As also shown, flexure F1, F1′ has essentially the opposite ratio asthat of flexures F2′ and F2″, i.e., flexure F1′ has a relatively highratio of y-direction stiffness (K_(Y1)), to x-direction stiffness(K_(X1)). It is also noted that while flexure F1, F1′, provides for thedesired x-direction oscillation, the instant inventors have alsodiscovered that a slightly arcuate path, such as shown in FIG. 6, tendsto improve the surface quality of the section. Thus, particularembodiments of flexure F1, F1′ may be provided with a ratio of length(L) to width (W) within the range of about 1.5 to 3, with a length Lwithin the range of about 5 to 12 cm.

Referring now to Table 1 below, the microtomes of the present invention,including flexures F2′, F2″, in combination with the low mass bladeholder 24′, have been shown to provide operational speeds of above 200Hz with parasitic z-motion of less than 2 microns, with particularembodiments exhibiting operational speeds of up to 300 Hz with less than1 micron of parasitic z-axis motion.

TABLE 1 Inventive Microtome Example Tissue: Rabbit myocardial, mousebrain Microtome Specifications Feed rate 0.1-10 mm/s Vibration Frequency15-400 Hz Vibration Amplitude 0.1-2 mm Section Thickness 10-2000 μmBlade angle 5-30 degrees Parasitic z-motion <1.5-3 μm CuttingCharacteristics Surface Quality <2 μm RMS

The flexures of this invention may be fabricated from substantially anymaterial. Prototypes have been fabricated using metallic materials, suchas aluminum, using an abrasive water jet cutting tool. Metalliccompliant mechanisms may be advantageous in that they may provide forboth elastic and plastic deformation of the mechanism's components. Itis further envisioned that the compliant mechanisms of this inventionmay be fabricated from other materials, such as silicon or doped siliconwafers, using a technique such as deep reactive ion etching (DRIE).

Having described various embodiments of the apparatus of the presentinvention, a representative method of operation will now be described.For example, a method of treating a sample includes using any of themicrotomes 20, 20′, 20″ to oscillate the microtome blade in a directiontransverse to a direction of advancing a cut; and moving the sample inthe cut direction, into operative engagement with the oscillatingmicrotome blade to remove a section from the sample. Optionally, thesample may be moved into operative engagement with an imaging device,such as a microscope, which is then used to image the sample. Inparticular embodiments, the image may be captured by the imaging deviceand stored for later reference.

The artisan of ordinary skill will readily recognize that there are manyvariable shapes and configurations for the various portions of theflexures that may be used to alter the various operational parameters ofembodiments of this invention. It should be further understood that anyof the features described with respect to one of the embodimentsdescribed herein may be similarly applied to any of the otherembodiments described herein without departing from the scope of thepresent invention.

In the preceding specification, the invention has been described withreference to specific exemplary embodiments for the purposes ofillustration and description. It is not intended to be exhaustive or tolimit the invention to the precise form disclosed. Many modificationsand variations are possible in light of this disclosure. It is intendedthat the scope of the invention be limited not by this detaileddescription, but rather by the claims appended hereto.

What is claimed is:
 1. A microtome apparatus comprising: a microtomeblade configured to oscillate in a direction transverse to a directionof advancing a cut; a first flexure configured to support and guide theblade, said first flexure configured to be compliant in the transversedirection while being stiff in the cut direction; a second flexureoperatively engaged at one end portion thereof with the first flexure,the second flexure configured for being stiff in the transversedirection while being compliant in the cut direction; a rotatingactuator; an eccentric driven by the actuator; the second flexure beingrotatably engaged at another end portion thereof with the eccentric,wherein operation of the actuator serves to oscillate the blade in thetransverse direction while effectively isolating non-transverse motionfrom the blade; and the second flexure being configured to moveindependently of any stationary objects during said oscillation; whereinthe first flexure is configured for being stiff in a direction (out ofplane direction) orthogonal to both the cut and transverse directions.2. The apparatus of claim 1, wherein the second flexure is configuredfor being stiff in the out of plane direction.
 3. The apparatus of claim1, wherein the blade is configured for oscillation at a frequency withina range of 15 to at least 400 Hz, an amplitude of movement in thetransverse direction within a range of 0.1 to at least 2 mm, and anamplitude of parasitic movement in the out of plane direction of no morethan 1.0 to 3.0 micron.
 4. The apparatus of claim 1, wherein the secondflexure is configured to move in a substantially frictionless mannerduring said oscillation.
 5. The apparatus of claim 4, wherein the secondflexure is monolithic.
 6. The apparatus of claim 5, wherein the secondflexure comprises a plurality of flexible beams spaced from one anotherin the cut direction.
 7. The apparatus of claim 6, wherein the pluralityof flexible beams extend from the one end portion towards the other endportion.
 8. The apparatus of claim 7, wherein the plurality of flexiblebeams extend substantially parallel to one another.
 9. The apparatus ofclaim 7, wherein the plurality of flexible beams are configured toextend in the transverse direction during a portion of said oscillation.10. The apparatus of claim 6, wherein during said oscillation, the oneend portion is configured to move in the transverse direction, whilebeing substantially prevented from moving in the cut direction, whilethe other end portion is configured to move in both the transverse andcut directions.
 11. The apparatus of claim 6, wherein the second flexurefurther comprises at least one connector disposed to join individualones of said flexible beams to one another, said at least one connectordisposed in spaced relation from said end portions.
 12. The apparatus ofclaim 11, wherein said at least one connector is disposed within themiddle 50 percent of the length L of the second flexure.
 13. Theapparatus of claim 1, wherein the second flexure has a ratioK_(x2)/K_(y2) of coefficient of stiffness in the transverse direction(K_(x2)) to coefficient of stiffness in the cut direction (K_(y2)) of atleast about 500 to
 2500. 14. The apparatus of claim 13, wherein themicrotome is configured to have a resonance frequency on the range of 2to 4 times the speed of oscillation in the transverse direction.
 15. Theapparatus of claim 13, wherein the microtome is configured to have aresonance frequency of at least 500 Hz.
 16. The apparatus of claim 2,wherein the microtome is configured to have an out-of-plane mode shapefrequency in the range of 4 to 8 times the speed of oscillation in thetransverse direction.
 17. The apparatus of claim 16, wherein themicrotome is configured to have an out-of-plane mode shape frequency ofat least 700 Hz.
 18. The apparatus of claim 1, wherein the first flexurehas ratio of length (L) in the cut direction to width (W) in thetransverse direction, within the range of about 1.5 to
 3. 19. Theapparatus of claim 1, wherein at least one of the first and secondflexures comprise a monolithic structure.
 20. The apparatus of claim 19,wherein said first and second flexures each comprise a monolithicstructure.
 21. The apparatus of claim 20, wherein said first and secondflexures comprise a single monolith.
 22. The apparatus of claim 1,wherein the blade is disposed on a blade holder, said blade holderextending in a cantilevered manner from the first flexure, the bladeholder having a center of rotation about an axis parallel to the cutdirection, said center of rotation being disposed on a transverse axispassing through said first and second flexures.
 23. The apparatus ofclaim 22, wherein said blade holder comprises a magnetic mountconfigured to retain the blade thereon.
 24. The apparatus of claim 1wherein at least one of said first and second flexures is fabricatedfrom an aluminum alloy.
 25. The apparatus of claim 1 wherein at leastone of said first and second flexures is fabricated from at least one ofaluminum, titanium, spring steel, or silicon.
 26. A tissue scanningimaging apparatus comprising: the microtome apparatus of claim 1; animaging device; and a stage configured to selectively advance a specimensample into operative engagement with both the microtome apparatus andthe imaging device.
 27. A method of treating a sample, said methodcomprising, with the microtome apparatus of claim 1: (a) oscillating themicrotome blade in a direction transverse to a direction of advancing acut; and (b) moving the sample in the cut direction, into operativeengagement with the oscillating microtome blade to remove a section fromthe sample.
 28. The method of claim 27, further comprising: (c) movingthe sample into operative engagement with an imaging device; and (d)imaging the sample.